Currently accepted therapies for most tumors are surgery, chemotherapy, radiation therapy, bone marrow transplants or various combinations of these therapies. In general these treatments are aimed at the destruction of the tumor cells by mechanisms independent of activation of the patient's immune system. In the course of radiation and chemotherapy significant damage to the immune system is an unfortunate side effect. Moreover, the long term effectiveness of these treatments for some tumors is questionable.
a. Activation of the Immune System
Within the last decade therapeutic approaches have been developed based on the activation of the immune system to mediate anti-tumor activity. Generally, a normal host response to tumor cells begins with T-cell recognition of tumor associated antigens on tumor cells or via antigen presenting cells. Recognition via T-cell antigen receptor triggers signal transduction pathways that mediate the activation of the T-cells. This results in secretion of interleukin-2 (IL-2), gamma-interferon, tumor necrosis factor-alpha, and other cytokines from the T-cells and accessory cells. The host immune system is thus mobilized to kill the tumor cells. However, for reasons that are poorly understood, for many tumors this host response does not occur or is inadequate to kill tumor cells.
One therapy designed to activate the immune system is the systemic administration of IL-2. However, the doses of IL-2 required to achieve adequate amplification proved to be toxic to the patient. Cellular immunotherapy approaches to activate the immune system have focused on two types of cells: LAK cells and TIL cells. LAK (lymphokine activated killer) cells are cells of the immune system which have been non-specifically activated through the use of cytokines such as IL-2 (Lotze et al., Cancer Res. 41, p. 4420-4425 (1981); Grimm et al., J. Exp. Med. 155, p. 1823-1844 (1982)) and/or the use of monoclonal antibodies such as anti-CD3 (Ochoa et al., Cancer Res. 49, p. 693-700 (1989)). These cells can mediate significant anti-tumor activity without the major histocompatibility complex (MHC) related restrictions characteristic of the T-cell receptor of classical cytolytic T-cells (CTL). In one recent study continuous infusion of IL-2 and LAK cells for advanced tumors resulted in responses in 12% of patients with melanoma and 3% of patients with renal cell carcinoma (Lotze, Cell Transplantation 2, p. 33-47 (1993)). While most LAK cells characterized to date consist of activated natural killer (NK) cells (Ortaldo et al., J. Exp. Med. 164, p. 1193-1205 (1986); Ferrini et al., J. Immunol. 138, p. 1297-1302 (1987); Phillips and Lanier, J. Exp. Med 164, p. 814-825 (1986)), LAK cells can also be generated from a subset of T-cells known as .gamma..delta. T-cells (T-cells which lack the classical .alpha..beta. subunits of the T-cell receptor and instead express the .gamma..delta. subunits) (Ochoa et al., Cancer Res. 49, p. 693-700 (1989)). LAK cells can also be generated from isolated CD4+ or CD8+ T-cells which have been cultured in the presence of IL-2 and anti-CD3 monoclonal antibodies (Geller et al., J. Immunol. 146, p. 3280-3288 (1991)). TIL (tumor infiltrating lymphocytes) cells are lymphocytes which have been isolated in vitro from tumors. Like LAK cells, these cells can be expanded by culturing in the presence of cytokines such as IL-2 or IL4, but, unlike LAK cells, these cells are tumor specific. Using TIL cells, a 20-50% response rate has been observed in patients with melanoma (Lotze, Cell Transplantation 2, p. 33-47 (1993)).
b. Enhancing Immunogenicity of Tumors
Other approaches for tumor immunotherapy involve increasing the immunogenicity of tumor cells rather than enhancing the activity of responding lymphocytes. It is believed that many tumor cells lack a degree of immunogenicity required to induce an adequate immune response (Houghton and Lewis, "Cytokine Induced Tumor Immunogenicity," ed. Forni et al., Academic Press, p. 35-54 (1994)).
Generally, stimulator cells (such as tumor cells) activate T-cells by engagement of the T-cell receptor with peptide associated with either Class I or Class II MHC molecules on the stimulator cell. These peptides can either be taken up from the external environment by the stimulator cell, in which case they are processed and presented along with MHC Class II molecules or, they can be peptides produced endogenously by the stimulator cell and then presented with MHC Class I molecules. Presentation of exogenously derived peptides by the stimulator cell is referred to as indirect presentation since the peptides are not being presented on the cell from which they were derived. In the case of direct presentation, peptides are presented on the surface of the cells from which the peptides were derived. FIG. 1 is a schematic diagram showing direct vs. indirect presentation.
In addition to the T-cell receptor and MHC antigens, a number of cell surface antigens have been identified that may play a role in mediating interactions between antigen presenting cells and the responder T-cells. These co-stimulatory molecules include intercellular adhesion molecules (ICAMs), vascular cell adhesion molecule 1 (VCAM-1), lymphocyte function-associated antigen 3 (LFA-3), heat stable antigen (HSA) and CD28 on lymphocytes, and the ligand B7 which must be present on the antigen presenting cell (Pardi et al., Immunol. Today 13, p. 224-230 (1992); Chen et al., Immunol. Today 14, p. 483-486 (1993)). Engagement of the T-cell receptor with the antigen presenting cell in the absence of costimulatory molecules can lead to T-cell anergy and failure of the immune response against the tumor (Gimmi et al., Proc. Natl. Acad. Sci. 90, p. 6586-6590 (1993)).
Unique tumor antigens have been defined for several tumors including the MAGE (van der Bruggen et al., Science 254, p. 1643-1650 (1991)) and MART (Kawakami, Proc. Natl. Acad. Sci. USA 91, p. 3515-3519 (1994); Boon et al., Ann. Rev Immunol. 12, p. 337-365 (1994)) antigens for melanoma and mucins for breast and pancreatic tumors (Finn, J. Cellular Biochem. 17D, p. 92 (1993); Domenech et al., J. Cellular Biochem 17D, p. 108 (1993); Fontenot et al., J. Cellular Biochem. 17D, p. 125 (1993)). See also Brown, J. P. et al., U.S. Pat. No. 5,141,742 (melanoma associated antigen). It has been observed that tumor cells do not efficiently present self-peptides (direct presentation) even when the cells do express MHC antigens, suggesting that there might be a defect/deficiency in another molecule necessary for effective direct presentation of antigen by tumor cells. Many tumor cells have been demonstrated to express low levels of B7. Accordingly, one therapeutic approach is to restore the immunogenicity of the tumor cells by the introduction of the gene for B7 into the patient's tumor cells, thus promoting direct tumor antigen presentation (Chen et al., Cell 71, p. 1093-1102 (1993) and EPO 600591; Chen et al., J. Exp. Med. 179, p. 523-532 (1994); Townsend and Alison, Science 259, p. 368-370 (1993); Baskar et al., Proc. Natl. Acad. Sci. USA 90, p. 5687-5690 (1993)). The introduction of the CD28 ligand B7 to immunogenic lymphoma, mastocytoma, melanoma or sarcoma resulted in increased CTL activity against the wild type tumor and protection against subsequent injection with the wild type tumor (Chen et al., Cell 71, p. 1093-1102 (1993) (melanoma); Chen et al., J. Exp. Med. 179, p. 523-532 (1994) (mastocytoma, fibrosarcoma, lymphoma, melanoma, carcinoma); Townsend and Alison, Science 259, p. 368-370 (1993); Baskar et al., Proc. Natl. Acad. Sci. USA 90, p. 5687-5690 (1993) (sarcoma)). Further, injection of EL4 lymphoma cells expressing B7 resulted in a 60% cure rate in mice with established EL4 derived tumors (Chen et al., J. Exp. Med. 179, p. 523-532 (1994)). Similarly, transfection of murine colon adenosarcoma or fibrosarcoma with genes for murine Class I molecules could mediate the regression of unmodified tumor, although tumors were not completely eliminated (Plautz et al., Proc. Natl. Acad. Sci. USA 90, p. 4645-4649 (1993) (fibrosarcoma, colon carcinoma)). This approach is currently being tested in human clinical trials (Nabel, Proc. Natl. Acad. Sci. USA 90, p. 94-97 (1993) (melanoma)). In both these examples the introduction of the foreign genes enhanced direct, Class I mediated recognition of the tumor cells by the effector cells of the host. The response is tumor specific. Treatment with the genetically modified tumor had no effect on the growth of an unrelated tumor. This response is throught to require direct cell-cell contact. See also Hock et al., Gene Therapy Weekly, p. 22 (Jan. 9, 1995) (murine neuroblastoma cells expressing Class II MHC).
A slightly different approach was taken by Trojan et al. for treatment of glioblastoma. Glioma cells express high levels of insulin-like growth factor I (IGF-1). Treatment of glioma cells with an anti-sense gene for IGF-1 appears to reverse the tumorogenic phenotype rendering the cells immunogenic. In these studies, injection of glioma cells expressing the IGF-1 anti-sense sequence resulted in elimination of pre-existing tumor in all animals treated (Trojan et al., Science 259, p. 94-97 (1993)). Although this response was shown to be mediated by CD8+ T-cells, it is not clear whether they are activated directly by the modified tumor cells or indirectly via antigens picked up by antigen presenting cells or both.
Additional approaches for enhancing the immunogenicity of tumors involve engineering the tumor cells to express cytokine genes such as IL-2, IL-4, IL-6, tumor necrosis factor, interferon-.gamma. or granulocyte macrophage colony stimulating factor (GM-CSF) (Dranoff et al., Proc. Natl. Acad. Sci. USA 90, p. 3539-3543 (1993); Golumbek et al., Science 254, p. 713-716 (1991) (renal cell carcinoma); Gansbacher et al., Cancer Res. 50, p. 7820-7825 (1990); Gansbacher et al., J. Exp. Med. 172, p. 1217-1224 (1990); Bannerji et al., J. Immunol. 152, p. 2324-2332 (1994) (fibrosarcoma); Fearon et al., Cell 60, p. 397-403 (1990) (colon carcinoma); Columbo et al., J. Exp. Med. 173, p. 889-897 (1991) (adenocarcinoma); Haddada et al., Hum. Gene Therapy 4, p. 703-711 (1993) (mastocytoma); Lollini et al., Int. J. Cancer 55, p. 320-329 (1993) (mammary adenocarcinoma); Watanabe et al., Proc. Natl. Acad. Sci. USA 86, p. 9456-9460 (1989) (neuroblastoma); Pardoll, Curr. Opin. Oncol. 4, p. 1124-1129 (1992); Tepper and Mule, Hum. Gene Therapy 5, p. 153-164 (1994); Porgador et al., Cancer Res. 52, p. 3679-3686 (1992) (Lewis lung carcinoma); See also WO 92/05262 Hopkins/University of Texas. Here too, the genetically modified tumor cells are able to stimulate an immune response in situations in which the parent tumor lines are non-immunogenic. Researchers in this area have observed that the immune response extends to destruction of unmodified tumor cells as well as the engineered tumor cells and can, in some cases result in complete regression of pre-existing tumor in experimental animals (Dranoff et al., Proc. Natl. Acad. Sci. USA 90, p. 3539-3543 (1993); Golumbek et al., Science 254, p. 713-716 (1991); Gansbacher et al., Cancer Res. 50, p. 7820-7825 (1990); Gansbacher et al., J. Exp. Med. 172, p. 1217-1224 (1990); Bannerji et al., J. Immunol. 152, p. 2324-2332 (1994) (fibrosarcoma); Fearon et al., Cell 60, p. 397-403 (1990); Columbo et al., J. Exp. Med. 173, p. 889-897 (1991); Haddada et al., Hum. Gene Therapy 4, p. 703-711 (1993); Lollini et al., Int. J. Cancer 55, p. 320-329 (1993); Watanabe et al., Proc. Natl. Acad. Sci. USA 86, p. 9456-9460 (1989); Pardoll, Curr. Opin. Oncol. 4, p. 1124-1129 (1992); Tepper and Mule, Hum. Gene Therapy 5, p. 153-164 (1994); Porgador et al., Cancer Res. 52, p. 3679-3686 (1992); Vieweg et al., Gene Therapy Weekly, p. 20 (Nov. 21, 1994) (prostrate cancer)).
In general these experimental protocols involve immunizing animals one or more times with irradiated tumor cells that have been genetically engineered to express the exogenous gene. Irradiation prevents the cells from dividing but does not diminish their antigenicity. Anti-tumor responses are then tested in one of three ways: (i) the animals are challenged with unmodified tumor cells after the immunization process is complete; (ii) the animals are challenged with unmodified tumor cells during the vaccination process; or (iii) small tumors are established before immunization with modified tumor cells.
The majority of the studies utilizing genetically modified tumor cells have involved the introduction of cytokine genes into various tumors (see Pardoll, Curr. Opin. Oncol. 4, p. 1124-1129 (1992); Tepper and Mule, Hum. Gene Therapy 5, p. 153-164 (1994) for reviews). One of the most effective molecules is GM-CSF (granulocyte macrophage-colony stimulating factor) which augments specific immunity for several tumor types (Dranoff et al., Proc. Natl. Acad. Sci. USA 90, p. 3539-3543 (1993) (B16 melanoma, colon carcinoma, lung carcinoma, fibrosarcoma, renal carcinoma)). GM-CSF is unique in that it may be mediating this anti-tumor effect by stimulating the proliferation and differentiation of dendritic cells which are extremely potent antigen presenting cells capable of presenting antigens to both CD4+ and CD8+ T-cells (Steinman, Ann. Rev. Immunol. 9, p. 271-296 (1991)). Metzinger has recently suggested that the only way the immune system can be activated to respond to tumors is via shed antigens being picked up and presented by professional antigen presenting cells such as dendritic cells (Metzinger, Ann. Rev. Immunol. 12, p. 991-1045 (1994)). Similarly Bannerji et al. have recently suggested that the rejection of IL-2 secreting fibrosarcoma cells is not T-cell mediated although the subsequent systemic immunity is dependent upon the presence of both CD4+ and CD8+ T-cells (Bannerji et al., J. Immunol. 152, p. 2324-2332 (1994)). They hypothesize that the destruction of the modified cells is mediated by NK cells resulting in the release of tumor antigens which can be taken up by antigen presenting cells expressing both Class I and Class II molecules on their cell surface. These cells would then be capable of activating both CD4+ and CD8+ T-cells. A similar model has been discussed by Shoskes and Wood (Shoskes and Wood, Immunol. today 15, p. 32-38 (1994)).
In recent experiments Cohen and co-workers have been able to prolong survival of mice with pre-existing melanoma by injecting the animals with allogeneic fibroblasts which have been transfected with the gene for IL-2 and DNA isolated from melanoma cells (Kim and Cohen, Cancer Res. 54, p. 2531-2535 (1994)). By using allogeneic cells there is no need to irradiate the cells, which could affect cytokine expression. Since the transfected cells are fibroblasts they do not form tumors and since they are allogeneic they readily activate the immune system. However, since they are readily rejected there is no long term stimulation of the immune system. Others have mixed cytokine expressing fibroblasts with irradiated tumor cells and then administered the mixture as a vaccine (WO 93/07906, PCT US92/08999). Still others have coupled nontumorous fibroblast cells to an adjuvant and administered the cells as a tumor vaccine (Eggers, U.S. Pat. No. 5,208,022).
Yet another therapy for prevention and treatment of tumors is immunization with tumor antigens (WO 93/06867 Pardoll, Mulligan). Another vaccine protocol is administration of irradiated tumor cells together with a bacterial adjuvant (Pardoll, 5 Cur. Opin. Immunology, p. 719-725 (1993). Others have irradiated unmodified tumor cells and administered them alone as a vaccine (Dranoff et al., 90 PNAS, p. 3539-3543, FIG. 4A (1993)).
c. Tumor Evolution
Most cancers are believed to be clonal in origin and that new subpopulations arise continuously during evolution of a cancer due to Darwinian selection of genetic variants that have a growth advantage. Some of the genetic variants are characteristic of a particular tumor type and in fact can serve as the basis for classifying the severity of tumor, in other cases the changes are idiotypic, i.e. specific to the individual's own tumor. Mutations giving rise to growth advantage include mutations in growth regulatory genes, changes in morphology, hormone dependence, enzyme patterns, and surface antigens. Some of these changes may allow the abnormal cells to escape either homeostatic controls of the patient or destruction by treatment. Conventional chemotherapies are often effective initially in slowing the progression of disease. However, with time, repeated treatments become less effective, perhaps through evolution of successively less sensitive clones (G. Klein and E. Klein, PNAS USA 74, p. 2121 (1977)). See also Schreiber, H., "Tumor Immunology," Chapter 32 in Fundatmental Immunology W. Paul, ed. (1993).
d. Diffusion Chambers
Diffusion chambers which prevent cell to cell contact have been used for many years to study immunologic mechanisms. Klein et al. have used tumor cells in a diffusion chamber as a model to study the host immune response to tumor cells. They conclude that tumor cells produce soluble factors that promote delayed type hypersensitivity and also stimulate angiogenesis which promotes tumor growth (Tumor Biol., 15, p. 160-165 (1994)).
Stillstrom has implanted tumor cells in diffusion chambers in order to induce immunity in rodents and found that after ten weeks the level of immunity induced by tumor cells in a diffusion chamber deposited subcutaneously for seven days was 10-100 times lower than that achieved with directly inoculated cells. In other experiments he found no significant difference in the immune state of animals inoculated directly and those given diffusion chambers containing tumor cells. He also found that chambers were rejected if left subcutaneously for several weeks (Acta Path. Microbiol. Scand., Sect. B 82, p. 676-686 (1974)).
Biggs and Eiselein used diffusion chambers to show that a certain tumor cell type releases a viral particle which diffuses out of the chamber providing immunity to subsequent challenge with the tumor cells. They also show that very low porosity of the chamber can prevent immunization (Cancer Research, Vol 25, p. 1888-1893 (1965)).
Cochrum et al. U.S. Pat. No. 5,015,476 discloses the use of lymphokines or cytokines as an adjuvant when micro-encapsulating parasites and implanting them to obtain immunization against parasitic infection.